weighed and recorded to measure average daily gain (ADG), average daily feed
intake (ADFI), and ratio between ADG and ADFI (G:F) of pigs. Diarrhea of each
pig was checked and its visual score was recorded by 3 independent evaluators
with a score from 1 to 5 (1 = normal hard feces; 2 = slightly soft feces; 3 =
soft, partially formed feces; 4 = loose, semi-liquid feces; and 5 = watery,
mucous-like feces) each day for the first 2 weeks of this experiment [16 (link)]. Frequency of diarrhea was calculated
by counting pen days with average diarrhea score from individual pigs in each
pen of 4 or greater [17 (link),18 (link)]. Whole blood samples were collected
from the jugular vein of randomly selected 2 pigs in each pen using EDTA tubes
(Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) containing
anticoagulant on day 1, 3, 7, and 14 after weaning. For the last week of the
experiment period, pigs were fed respective dietary treatments containing 0.2%
chromic oxide as an indigestible marker. Fecal samples were collected from
randomly selected 2 pigs in each pen by rectal palpation daily for the last 3
days after the 4-d adjustment period. The collected fecal samples were pooled
and stored at −20°C until analysis. Diet samples were also
collected and stored at −20°C until analysis. Randomly selected 2
pigs in each pen were anesthetized by an intra-muscular injection of a 2-mL
suxamethonium chloride (Succicholine®, Ilsung Pharm. Co. Ltd.,
Seoul, Korea) at the end of this experiment. After anesthesia, pigs were
euthanized by CO2 gas [19 (link)].
Ileal digesta samples were collected from distal ileum before the ileocecal
junction [11 ]. The collected ileal
digesta samples were stored at −20°C until analysis. Sections of
ileum of 3 cm length were collected, washed gently with distilled water, and
fixed in 10% neutral buffered formalin for histological analysis [20 (link),21 (link)].